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. 2024 Mar 27;27(4):109591. doi: 10.1016/j.isci.2024.109591

Figure 4.

Figure 4

Alexidine analogs induce mitochondrial bioenergetic stress

(A) H1299 cells were treated with 1 μM of the indicated analogs for 48 h. Mitochondrial membrane potential was detected by flow cytometry using TMRM and mitochondrial amount was detected in parallel using MitoTracker Green. Representative histograms normalized to the mode are shown.

(B) Cells were treated as in (A). At the time of TMRM staining, FCCP was added to the samples and cells were analyzed by flow cytometry. Representative histograms normalized to the mode are shown.

(C) Cells were treated as in (A). At the time of TMRM staining, oligomycin was added to the samples and cells were analyzed by flow cytometry. The quantification of the TMRM-low population of three biological replicates is shown in the graph to the right. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used to determine significance.

(D) Cells were treated for 48 h with the analogs as indicated. Lysates were separated by western blot and probed for phosphorylated ubiquitin (pUb (S65)), total ubiquitin (Ub), SQSTM1, and actin. See Figure S4 for quantification of SQSTM1.

(E) Cells were treated with 1 μM of the indicated analogs for 48 h. The oxygen consumption rate (OCR) was detected over time by the Agilent Seahorse XF Analyzer with the addition of oligomycin (oligo), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and a rotenone/antimycin mixture at the indicated timepoints. A representative curve is shown.

(F) The basal respiration, maximal respiration, spare respiratory capacity, and ATP-linked respiration were calculated from the OCR in (E).

(G) Cells were treated with the indicated analogs for 48 h. The media was collected and lactate amount detected. Lactate concentration was normalized relative to cell count and DMSO control. Significance was assessed using an ordinary two-way ANOVA with a Tukey’s multiple comparisons test. For all experiments, three biological replicates were performed. Mean ± SD is shown. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.